The mammalian kidney is used as a model to study the genetic basis of development and oncogenesis in a complex multicellular tissue. Current studies continue to focus on the role of transcription factors in early kidney development and their regulation by potential tumor suppressor genes. Our major goals are to understand the molecular mechanisms through which developmental regulators affect complex morphogenetic pathways. Previously, we have determined that the Pax-2 gene is required for the conversion of early kidney mesenchymal cells to an epithelial phenotype, that the Pax-2 was persistently expressed in human embryonal kidney tumors, and that persistent expression in transgenic mice inhibits terminal differentiation of renal epithelial cells resulting in a condition similar to human nephrotic syndrome. More recently, progress has been made in several new areas. A. Development. Although Pax-2 is required for early kidney development, whether it is sufficient to initiate the conversion of mesenchymal cells to epithelium has not been determined. Thus, we designed a novel experiment using Pax-2 transducing retroviruses to introduce the active gene into the mesenchymal cells prior to endogenous Pax-2 activation. Preliminary results indicate that expression of Pax-2 is enough to convert the mesenchyme cells into an early epithelial like structure. These results suggest a master regulatory function for Pax-2 in the specification of epithelial cells in the kidney. B. Oncogenesis. Expression of Pax-2 is repressed during the course of normal renal epithelium development. However, in embryonal kidney carcinomas, such s Wilms' tumor, and in adult renal cell carcinoma Pax-2 expression levels are still high. We have been able to demonstrate that the suppression of Pax-2 during kidney development is mediated, in part, by the Wilms' tumor suppressor gene, WT1. There is a direct correlation between decreasing levels of Pax-2 protein and increasing levels of WT1 in the developing nephron. Furthermore, WT1 binds at least three sites in the Pax-2 5' regulatory sequences and can repress transcription in vitro. Thus, we have established a link between a developmental regulatory gene and a known tumor suppressor gene. In adult tumors, reactivation of Pax-2 may be a necessary determinant for oncogenesis. Inhibition of Pax-2 gene expression by antisense methods results in growth inhibition of renal cell carcinoma derived cell lines. These results suggest a function for Pax-2 in the proliferation of the early epithelial cell, a process concurrent with differentiation. C. Biochemistry. In order to understand the function of the Pax family of transcription factors, identifying the potential target genes is mandatory. Using Pax-2 specific antibodies, DNA-protein complexes were precipitated from intact chromatin and the DNA binding sequences cloned. DNA footprinting and methylation protection experiments clearly demonstrate Pax-2 binding to these genomic loci. These genomic tags can be used to identify larger genomic clones that may contain potential target genes.